LC-MS/MS for Quantitative Analysis

After a century of development, LC-MS/MS has become more and more widely used in various fields such as medicine, food, environment, forensic medicine, and clinical medicine. Especially suitable for clinical testing, the scope of application far exceeds the scope of radioimmunoassay and chemical testing, which is unmatched by other methods.

LC-MS/MS has the characteristics of high sensitivity, strong selectivity, and good accuracy. So, how many times is the liquid quality stronger than the liquid quality? Isn’t there any place for liquid quality? It seems unlikely. . .

Not much nonsense, today’s focus is to help you get a quantitative analysis of liquid quality!

Hold on, it doesn’t matter, follow these eight steps, do it a few more times, and always remember: What makes perfect!

The first step: Q1 SCAN——Determine the mass-to-charge ratio of the precursor ion

    According to the nature of the compound to be tested, select the polarity (+/-) and ionization method (ESI/APCI) of the analysis, and select the scanning range and time according to the molecular weight

    Determine the mass-to-charge ratio of the precursor ion, accurate to one decimal place.

parameter settings:

Scan mode: Q1 Scan; Mass range: Parameter Range, 100～MW+30, t=1～2sec, Center Width, MW±5Da, t=0.5sec; Manually adjust DP and GS1 to make the spraying stable and effective.

Step 2: Product Ion Scan —— Determine the mass-to-charge ratio of product ions

    Obtain high-quality MS2 images. The intensity of the precursor ion should be 1/3 to 1/4 of the intensity of the base peak in the spectrum.

    After smoothing, select the mass-to-charge ratio of the product ion and determine it to one decimal place

parameter settings

Scanning method: Product Ion Scan; quality range: Parameter Range, 50～MW+10, t=1～2sec;

Adjust the CE manually.

Step 3: Optimize compound parameters

    According to the previously selected parent and daughter ions, an MRM ion pair is formed

    When selecting multiple transitions, the analysis time of each pair should be allocated reasonably

    Use “Ramp” to optimize the parameters under the Compound item and the CAD parameters under the Source item. Each ion is set separately.

    Preliminary establishment of MRM method

  Generally optimize twice to get more accurate results

The fourth step: Method transformation: change continuous injection method → LC-MS method

    Turn off all mass spectrometry interfaces, inactivate the existing mass spectrometry system, and activate liquid chromatography and mass spectrometry equipment.

    Click Build Acquire Method in Acquire, open the MRM method saved above, and add equipment: chromatography pump, autosampler, etc.

    Set up chromatogram synchronization.

    Set the chromatographic parameters, the analysis time is generally 0.6~1min, the mobile phase composition and flow are the same as the actual sample analysis; modify the mass spectrometry time to be consistent with the chromatographic parameters; set the initial values of GS1, GS2, and TEM to around 40, 40, 400 Save method, for example: LC-MRM.dam.

Step 5: FIA-Optimize Source Parameters

    Dilute the previously used sample solution 100-1000 times.

    Under Tune, double-click Quantitative Optimization, select FIA analysis,

Automatically optimize according to the wizard. FIA quantitative optimization without connecting the column

    Note: The optional values that need to be optimized are separated by “;”.

    When the meaning and settings of the source parameters are clear, this step can be omitted.

Step 6: Optimization of chromatographic conditions

    Connect and balance the chromatographic column, use appropriate concentration standards to investigate the separation;

    According to the chromatographic separation situation, gradient elution will be set;

    In MRM mode, it is not necessary for all peaks to be baseline separated, but it should be avoided;

Interference of matrix ion suppression;

    Adjust the chromatographic mobile phase composition and flow rate, and adjust the source parameters accordingly.

Step 7: Making a standard working curve

    Add standards to the blank matrix and dilute to different concentrations;

    According to the composition of the LC mobile phase, select the diluent and prepare the actual sample;

    Edit batch files and arrange the order of sample injection;

    Balance the MS ion source and chromatographic column;

    LC-MS/MS collects data.

Step 8: Data processing

Preparation of standard curve, correct integration of chromatogram, calculation method with and without internal standard.

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